1. Field of the Invention
The present invention relates to a microRNA for the identification of exposure to lower aliphatic saturated aldehydes and a method for the identification thereof using the same, more precisely a microRNA whose expression is changed specifically by the treatment of aldehydes at the concentration demonstrating 80% cell survival rate and a method for the identification of exposure to such aldehydes using the same.
2. Description of the Related Art
Micro RNA (miR, miRNA) has been recently on the rise as the important regulative RNA affecting a variety of biological processes. These small non-coding RNAs (typically 18-24 nucleotides long) are able to accelerate RNA degradation, inhibit mRNA translation, and regulate protein expression pattern by affecting gene transcription. Micro RNA plays a key role in a variety of biological processes such as development and differentiation, cell proliferation control, stress response, and metabolism, etc. Approximately 1000 human micro RNAs are known so far.
Micro RNA is transcribed by RNA polymerase II (pol II) or RNA polymerase III (pol III; Qi, P. et al. Cell. Mol. Immunol. 3, 411-419, 2006), and can be induced from each microRNA gene, intron of a gene encoding protein, or poly-cistron transcripts encoding mainly mRNAs closely related to each other. Transcription of miRNA by RNA pol II or pol III produces the first transcript, which is so called primary miRNA transcript (pri-miRNAs) in the length of thousands of nucleotides. In nucleus, pri-miRNAs are processed by RNAse, Drosha, to produce 70-100 nucleotide long hairpin shaped pre-miRNAs. After being transferred in cytoplasm, hairpin pre-miRNA is additionally processed by dicer to produce double-stranded miRNA. Mature miRNA strand is mixed in RNA-induced silencing complex (RISC) for the binding with target mRNAs based on base pair complementarity. If miRNA base pairs are consistant with target mRNAs, which is very rare though, it accelerates the degradation of mRNA. More generally, miRNAs form heteroduplex with target mRNAs, which affects mRNA translation.
Micro RNA mechanism greatly affects cancer development, cellular aging, and organ growth, etc. Therefore, the micro RNA related studies not only play a key role in explaining casual relations of vital phenomena such as cancer development and aging, etc, but also can be applied to further studies in relation to development and differentiation and for the development of cell therapy products using stem cells, indicating those studies may be the core of future bio-studies in Korea. Therefore, the development of micro RNA marker is important since it can help early diagnosis and prediction of diseases including cancer.
Besides, micro RNA marker is expected to be effectively used for the prediction of exposure to a specific environmental hazardous substance. Studies have been focused so far on the investigation of gene (mRNA) changes caused by such environmental hazardous substances and the relation with disease thereby. However, according to the recent increase of interest in micro RNA, the expression changes of micro RNA caused by exposure to harmful substances such as benzene, arsenic or RDX, etc, have been investigated in recent studies and thereby the micro RNA whose expression is specifically changed by the said substances and target genes thereof have been proposed as markers for the harmful substances (Baccarelli, A. & Bollati, V. Curr. Opin. Prediatr. 21, 243-251, 2009). Micro RNA is believed to be an effective marker not only for the prediction of exposure but also for the prediction of toxic mechanism caused by environmental hazardous substances and an effective regulator controlling target gene expression as well. Although micro RNA marker is a key player in the prediction of exposure to environmental hazardous substance and toxic mechanism thereof, the studies on micro RNA have been limited to the development of a marker for diagnosis of disease and the studies on the expression changes of micro RNA caused by exposure to harmful substances such as aldehydes which are so easy to be exposed on various environments are still insufficient. Epigenetic changes are not as big as the diversity of genetic changes including gene expression. Therefore, compared with gene expression profiling requiring multiple markers, disease or harmful substance exposure can be early diagnosed with simple epigenetic marker such as micro RNA or DNA methylation. The said diagnosis can be performed by non-invasive methods. Each micro RNA regulates different target genes. Higher eukaryote has thousands of micro RNAs, suggesting that potential circuits that can be regulated by micro RNA seem to be very huge.
Among many aldehydes, lower aliphatic saturated aldehydes have C0˜C9 carbon chain. Each of them is gas or liquid having pungent odor and dissolved in water. Among them, aldehydes having C6˜C9 carbon chain are mainly used as flavorings or food additives, or used in perfume industry. The indoor or outdoor concentrations of lower aliphatic saturated aldehydes have not been fully investigated since 8 kinds of aldehydes except formaldehyde and acetaldehyde have not been classified as hazardous materials or targets for regulation, yet. According to the previous reports rarely made, the exposure level of lower aliphatic saturated aldehydes has not been regular. For example, the exposure level in indoor environment was varied from conditions of the building and surrounding environment. Only Korea and Japan have the regulation on lower aliphatic saturated aldehydes, which is exemplified by propionaldehyde, butylaldehyde, and pentylaldehyde (only three of them).
Once exposed to a low concentration of lower aliphatic saturated aldehyde, such symptoms as ocular irritation and respiratory irritation are developed. When a high concentration of the lower aliphatic saturated aldehyde is inhaled, respiratory system is irritated, resulting in such symptoms as burning feeling, nausea, dizziness, cough, phlegm, laryngitis, headache, respiratory rate increase, and dyspnea. When the said aldehyde is inhaled for a long term, convulsion, seizure, bronchitis, pneumonia, and laryngeal edema can be developed (www.toxnet.nlm.nih.gov). The in vivo function of lower aliphatic saturated aldehyde is to induce carbonylation of nucleic acid and protein to cause mutation or to induce oxidation to cause cytotoxicity (Crit Rev Toxicol 35(7):609-662, 2005).
The volatile organic compounds flowing through bloodstream affect the lung by diffusion of lung sac membrane. Hexane, methylpentan, isopropene, and benzene have been used as markers for the respiratory diseases (J Vet Sci 5(1):11-18, 2004). Recently, a simple diagnostic method for lung cancer has been developed based on the results of exhaled breath analysis on volatile organic compounds. Among the volatile organic compounds, aldehydes are the most representative materials commonly found in lung cancer patients (J Chromatogr B Analyt Technol Biomed Life Sci. 878(27):2643-2651, 2010). Therefore, the lower aliphatic saturated aldehyde specific biomarker can be effectively used for the screening of exposure to lower aliphatic saturated aldehyde in the environment along with the screening of pulmonary disease. Once exposed to aldehydes, bronchus related symptoms are mainly developed, which are cough, phlegm, laryngitis, nausea, asthma, and dizziness, etc. If it gets worse, such diseases as pneumonia, laryngeal edema, bronchitis, and seizure can be developed (www.toxnet.nlm.nih.gov).
Despite the hazard in human, risk assessment data of lower aliphatic saturated aldehyde are not enough. Recently, microarray chip is widely used for the diagnosis of diseases. However, the methods for the screening of exposure to environmental hazardous substance are limited to a few classical methods such as GC-MS (Gas Chromatography-Mass Spectrometer) or HPLC (High Performance Liquid Chromatography). GC-MS or HPLC enables quantitative analysis but proper conditions have to be set up first and expensive equipments are required for the analysis. Therefore, faster and simpler screening method, such as real-time RT-PCR using microarray chip or primer is more recommended to evaluate risk quickly. Thus, it is important to establish molecular index for the screening of toxicity, especially micro RNA expression pattern involved in various diseases including cancer via faster and simpler methods such as real-time RT-PCR (real-time reverse transcript polymerase chain reaction) using primers or microarray chip for the fast risk assessment, and so is to control and manage the exposure to lower aliphatic saturated aldehydes.
Ever since micro RNA was first identified in 1997, many kinds of micro RNAs have been rapidly identified from mammals and microorganisms, which were reported to Sanger miRBASE database (www.mirbase.org/index.shtml) Based on the micro RNA data established so far, genome-wide expression studies have been actively undergoing to disclose gene functions. Microarray assay is generally used to analyze the expressions of thousands of genes at a time (Schena, M, et al. Proc. Natl. Acad. Sci. USA 93, 10614-10619, 1996).
Microarray indicates the glass board on which many sets of cDNA (complementary DNA) or 20-25 base pair long oligonucleotides are integrated. cDNA microarray is now produced by ink jetting or by fixing cDNA mechanically on the chip in laboratories of schools or companies including Agilent and Genomic Solutions, etc. (Sellheyer K. et. al. J. Am. Acad. Dermatol. 51, 681-692, 2004). Oligonucleotide microarray is produced by direct synthesis on the chip using photolithography by Affymetrix Co., or via fixation of synthesized oligonucleotides by Agilent Co. (Sellheyer, K. et. al. J. Am. Acad. Dermatol. 51, 681-692, 2004).
To analyze gene expression, micro RNA is first obtained from samples such as tissues, etc., followed by hybridization with oligonucleotides on microarray. The obtained micro RNA is labeled with fluorescein or isotope.
The methods to analyze gene expression using microarray are largely divided into two ways; one is two-dye method and the other is one-dye method. When complementary binding is measured, the control and the experimental samples are labeled with different fluorescent materials (ex, Cye3 and Cye5), which proceed to the reaction with microarray. This is called two-dye microarray. If the control and the experimental samples are labeled with the same fluorescent material and then reacted with two different microarrays, it is called one-dye microarray (Vivian, G. et al. Nature 21, 15-19, 1999).
The cooperation with toxicogenomics, the most recent technology using DNA microarray, enables high throughput quantitative analysis and expression pattern analysis of micro RNAs expressed in a specific tissue or cell line triggered by every chemical including not only drugs and new drug candidates but also representative environmental contaminants. Thus, specific genes that are involved in side effects of drugs and adverse actions of environmental contaminants can be identified by analyzing specific micro RNA expression in specific cells. Accordingly, adverse actions of environmental contaminants and molecular mechanisms related to functions and side effects of drugs can be understood and further screening and identification of such material that causes toxicity and side effects can be achieved.
The present inventors observed and analyzed micro RNA expression profiles in A549, the human lung cancer cell derived cell line, treated with lower aliphatic saturated aldehydes at the concentration of IC20 (the concentration showing 20% survival rate) by using oligomicroarray on which 1347 human micro RNAs were integrated. As a result, the micro RNA showing changes of expression specifically by lower aliphatic saturated aldehyde was identified. Accordingly, the present inventors completed this invention by establishing the method for the identification of exposure to lower aliphatic saturated aldehyde by using the micro RNA.